Cardiovascular disease is the leading cause of death in the United States and hypertension is a major risk factor for the development of cardiovascular disease. Alarmingly, statistics indicate that more than a quarter of the US population had hypertension in 2004. Contemporary research has demonstrated that the caudal brain stem exerts a profound influence over cardiovascular function. In particular, neurons of the rostroventrolateral medulla (RVLM) are known to be an important node in a central network that contributes to this regulation and malfunction of the network in experimental animals can produce hypertension. This network involves multiple cell groups in the brain stem, midbrain, diencephalon and forebrain. We recently developed a novel technology that allowed us to map the projections of C1 catecholamine neurons in RVLM. The data from that investigation confirmed the projection of C1 neurons to the sympathetic column in thoracic spinal cord but also revealed dense projections to a number of supraspinal cell groups with demonstrated influences upon cardiovascular function. In this application we hypothesize that collateralization of the C1 population to cardiovascular regulatory cell groups provides the neural substrate to coordinate activity within this distributed network. Experiments in three Specific Aims are advanced to test this hypothesis. Aim 1 will use our novel technology (lentivirus mediated anterograde tracing of C1 projections) and electron microscopy to test the hypothesis that recurrent collaterals of C1 neurons synapse upon projection specific and phenotypically distinct populations of RVLM neurons. Lentivirus mediated reporter gene (EGFP) expression will be used to label C1 axon terminals and ultrastructural analysis will characterize the synaptology of these afferents with subpopulations of neurons that differ in phenotype, projection patterns and topography within RVLM. Aim 2 will test the hypothesis that reticulospinal C1 neurons provide a substrate for coordination of network activity through projections that collateralize diffusely to supraspinal targets. Although surprisingly few studies have addressed this issue it is widely accepted that reticulospinal C1 neurons do not contribute substantially to supraspinal projections. Nevertheless, our data and that derived from a small number of studies examining C1 efferents challenge this conclusion. Experiments in this aim will employ dual retrograde labeling combined with immunocytochemical characterization of neuronal phenotype to address this question in a comprehensive fashion. Aim 3 will utilize conditional replication of pseudorabies virus and transneuronal labeling to test the hypothesis that reticulospinal C1 neurons receive synaptic input from all sources of RVLM afferents. Importantly, we hypothesize that neuronal circuits synaptically linked to reticulospinal C1 neurons will constitute a subset of the neurons that project to RVLM. These experiments will exploit a novel technology for conditional replication of PRV. Lentivirus vectors will be injected into RVLM to achieve targeted expression of cre recombinase (CRE) in C1 neurons or in neurons throughout RVLM. A strain of pseudorabies virus (PRV- 2001) whose replication is conditional upon the presence of CRE will be injected into the projection targets of reticulospinal RVLM neurons in thoracic spinal cord. Retrograde transport of PRV-2001 to RVLM will lead to restricted replication of the virus in CRE-containing neurons and retrograde transneuronal labeling of synaptically linked neurons. Collectively these data promise to provide important functional insights into the way in which C1 and the RVLM contribute to cardiovascular homeostasis as well as providing a wiring diagram that should improve understanding of the neural basis of neurogenic hypertension. PUBLIC HEALTH RELEVANCE: A distributed network of neurons in the central nervous system is known to exert a regulatory influence over cardiovascular function and malfunction of the network can produce hypertension, a major risk factor for the development of cardiovascular disease. However, the way in which neuronal activity is coordinated within the central cardiovascular network is not known. This proposal tests the hypothesis that collateralized projections of C1 catecholamine neurons in the rostroventrolateral medulla provides the neural substrate for this integration.